A hybridoma cell line secreting chloronitrosamine monoclonal antibody and its application

Abstract

This invention relates to a hybridoma cell line secreting a monoclonal antibody against nitrosamine and its application, belonging to the field of immunochemistry. This invention utilizes 4-amino-3,5-dichlorobenzoic acid as a nitrosamine hapten to prepare a complete nitrosamine antigen. After cell fusion and screening using an indirect competitive enzyme-linked immunosorbent assay (ELISA), a hybridoma cell line is obtained. The monoclonal antibody against nitrosamine secreted by this cell line exhibits good detection sensitivity for nitrosamine, with an IC50 value of [missing value]. 50 With a value of 3.5 ng / mL, it can be used to establish an immunological detection method for chloronitrosamines and to detect chloronitrosamine residues in food.

Description

Technical Field

[0001] This invention relates to the field of immunochemical technology, and in particular to a hybridoma cell line that secretes a monoclonal antibody against chloronitrosamine and its application. Background Technology

[0002] 2,6-Dichloro-4-nitroaniline, also known as 2,6-dichloro-4-nitroaniline, began to be commercially sold as a fungicide in the late 1950s. As a broad-spectrum agricultural fungicide, it can control sclerotinia rot in sweet potatoes, kenaf, cucumbers, lettuce, cotton, tobacco, strawberries, and potatoes; soft rot in sweet potatoes, cotton, and peaches; late blight in potatoes and tomatoes; wilt in apricots, almonds, and apples; wheat smut; and flower rot in broad beans. 2,6-Dichloro-4-nitroaniline can be mixed with most insecticides, fungicides, Bordeaux mixture, and lime-sulfur mixtures.

[0003] For the detection of chloronitrosamine pesticide residues, high-performance liquid chromatography (HPLC), gas chromatography (GC), and GC-MS / MS are commonly used. However, these methods have drawbacks such as complex sample pretreatment and long detection times, making them unsuitable for rapid detection of large numbers of samples. To protect the interests of consumers, it is necessary to establish a highly efficient and rapid detection method for chloronitrosamine. Enzyme-linked immunosorbent assay (ELISA) is an extremely efficient, sensitive, and rapid detection method. It requires simple sample pretreatment, involves few purification steps, has a large analytical capacity, low detection cost, and is easy to operate, making it suitable for rapid on-site detection of large numbers of samples. Therefore, it has been widely used in pesticide residue analysis. However, the prerequisite for using ELISA to detect chloronitrosamine is obtaining a monoclonal antibody with high specificity and sensitivity to chloronitrosamine. Therefore, finding a method for preparing a monoclonal antibody with high specificity and sensitivity to chloronitrosamine is crucial. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a hybridoma cell line that secretes a monoclonal antibody against chloronitrosamine and its applications. The monoclonal antibody against chloronitrosamine secreted by the hybridoma cell line provided by this invention exhibits good specificity and detection sensitivity (IC50) for chloronitrosamine. 50 With a value of 3.5 ng / mL, it can be used to establish an immunological detection method for chloronitrosamines to detect chloronitrosamine residues in food.

[0005] The first objective of this invention is to provide a hybridoma cell line that secretes a monoclonal antibody against chlornitramine, which has been deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. It is classified as a monoclonal cell line, deposited on April 18, 2024, with accession number CGMCC No. 45917.

[0006] The second objective of this invention is to provide a method for preparing a hybridoma cell line that secretes chloronitrosamine monoclonal antibodies. The chloronitrosamine hapten used in the preparation process is 4-amino-3,5-dichlorobenzoic acid, with the following molecular structural formula:

[0007]

[0008] Furthermore, the complete chloronitrosamine antigen used in the preparation process is BJS-KLH, with the following molecular structure:

[0009]

[0010] Furthermore, the preparation method of chloronitrosamine complete antigen includes the following steps:

[0011] Weigh 4-amino-3,5-dichlorobenzoic acid and N-hydroxysuccinimide (NHS) and dissolve them in N,N-dimethylformamide (DMF). Stir at room temperature to obtain mixed solution A. Weigh 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, dissolve it completely in DMF, and add it to mixed solution A. Stir at room temperature for 4-6 hours to obtain mixed solution B. Take hemocyanin (KLH), dilute it to 3 mg / mL with carbonate buffer, and slowly add mixed solution B dropwise to it. React at room temperature overnight to obtain reaction solution. Then dialyze the reaction solution with phosphate buffer to remove unreacted small molecule haptens to obtain chloronitrosamine complete antigen BJS-KLH.

[0012] Furthermore, the method for preparing the chloronitrosamine-coated antigen includes the following steps:

[0013] 4-Amino-3,5-dichlorobenzoic acid and N-hydroxysuccinimide (NHS) were dissolved in anhydrous N,N-dimethylformamide (DMF) and reacted with stirring at room temperature to obtain mixed solution C. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride was dissolved in anhydrous N,N-dimethylformamide and added to mixed solution C. The mixture was stirred at room temperature for 4-6 hours to obtain mixed solution D. Chicken ovalbumin was diluted to 3 mg / mL with carbonate buffer (CBS), and then mixed solution D was slowly added dropwise to it to obtain the reaction solution. The reaction solution was dialyzed with PBS to remove unreacted small molecule haptens, yielding chloronitrosamine-coated antigen (BJS-OVA).

[0014] Furthermore, the method for preparing a hybridoma cell line secreting chloronitrosamine monoclonal antibodies includes the following steps:

[0015] Step S1: Prepare nitramine complete antigen using nitramine hapten; mix the obtained nitramine complete antigen with complete Freund's adjuvant to obtain antigen-containing complete Freund's adjuvant; mix the obtained nitramine complete antigen with incomplete Freund's adjuvant to obtain antigen-containing incomplete Freund's adjuvant.

[0016] Step S2: Subcutaneously immunize the immunized animals with the complete Freund's adjuvant containing the antigen obtained in step S1;

[0017] Step S3: The immunized animals in Step S2 are given a booster immunization with the incomplete Freund's adjuvant containing antigen obtained in Step S1, and then immunized with chlornitramine complete antigen via sprint immunization.

[0018] Step S4: Take spleen cells and myeloma cells from the immunized animals that underwent sprint immunization in step S3 and perform cell fusion.

[0019] Step S5: Use indirect ELISA to screen out positive cell wells, select chloronitramine as a standard, use indirect ELISA to determine the inhibitory effect on positive cells, use limiting dilution method for subcloning, and finally screen out the hybridoma cell line that secretes chloronitramine monoclonal antibody.

[0020] A third objective of this invention is to provide a chloronitrosamine monoclonal antibody secreted by the aforementioned hybridoma cell line.

[0021] Furthermore, the preparation method of chloronitrosamine monoclonal antibody includes the following steps:

[0022] Take 8-10 week old BALB / c mice, and inject each mouse intraperitoneally with 1 mL of sterile paraffin oil. Seven days later, inject each mouse intraperitoneally with 1×10 6 Ascites fluid was collected from chloronitrosamine hybridoma cells starting from day 7. The ascites fluid was purified for antibody purification using the caprylic acid-saturated ammonium sulfate method. The purified monoclonal antibody was stored at -20°C.

[0023] A fourth object of the present invention is to provide a composition comprising the above-described hybridoma cell line and / or the above-described chloronitrosamine monoclonal antibody.

[0024] A fifth objective of the present invention is to provide a test strip containing the above-mentioned hybridoma cell line, the above-mentioned chlornitramine monoclonal antibody, or the above-mentioned composition.

[0025] A sixth object of the present invention is to provide a kit containing the above-described hybridoma cell line, the above-described chloronitrosamine monoclonal antibody, or the above-described composition.

[0026] A seventh object of the present invention is to provide a chip containing the above-mentioned hybridoma cell line, the above-mentioned chloronitrosamine monoclonal antibody, or the above-mentioned composition.

[0027] An eighth object of the present invention is to provide the application of the above-described chloronitrosamine monoclonal antibody, the above-described composition, the above-described test strip, the above-described kit, or the above-described chip in the detection of chloronitrosamine.

[0028] The technical solution of the present invention has the following advantages compared with the prior art:

[0029] The monoclonal antibody against nitrosamine secreted by the hybridoma cell line described in this invention exhibits good specificity and detection sensitivity (IC50) for nitrosamine. 50 With a value of 3.5 ng / mL, it can be used to establish an immunological detection method for chloronitrosamines to detect chloronitrosamine residues in food.

[0030] Preservation of biological materials

[0031] The hybridoma cell line secreting chlornitrosamine monoclonal antibody is deposited at the China General Microbiological Culture Collection Center, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. It is classified as a monoclonal cell line, deposited on April 18, 2024, with accession number CGMCC No. 45917. Attached Figure Description

[0032] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein...

[0033] Figure 1 This is the standard curve of the inhibition of chloronitrosamine by the monoclonal antibody of the present invention. Detailed Implementation

[0034] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0035] The culture media involved in the examples are as follows:

[0036] RPMI-1640 medium (mg / L): L-arginine 290, L-asparagine 50, L-aspartic acid 20, L-cysteine ​​dihydrochloride 65.15, L-glutamic acid 20, glycine 10, L-histidine 15, L-hydroxyproline 20, L-isoleucine 50, L-leucine 50, L-lysine hydrochloride 40, L-methionine 15, L-phenylalanine 15, L-proline 20, L-serine 30, L-threonine 20, L-tryptophan 5, L-tyrosine 23. 19. L-Valine 20. Para-aminobenzoic acid 1. Calcium nitrate 100. Anhydrous magnesium sulfate 48.84. Anhydrous sodium dihydrogen phosphate 676.13. Potassium chloride 400. Sodium chloride 6000. Glucose 2000. Reduced glutathione 1. Phenol red 5. L-glutamine 300. Biotin 0.2. D-calcium pantothenate 0.25. Folic acid 1. I-inositol 35. Nicotinamide 1. Choline chloride 3. Pyridoxine hydrochloride 1. Riboflavin 0.2. Thiamine hydrochloride 1. Vitamin B12 0.005. Sodium bicarbonate 2000.

[0037] The reagents involved in the following examples are as follows:

[0038] Carbonate buffer (CBS): Weigh 1.59 g of Na2CO3 and 2.93 g of NaHCO3, dissolve them separately in a small amount of double-distilled water and mix them together. Add double-distilled water to about 800 mL and mix well. Adjust the pH to 9.6 and add double-distilled water to a final volume of 1000 mL. Store at 4°C for later use.

[0039] Phosphate-buffered saline (PBS): 8.00g NaCl, 0.2g KCl, 0.2g KH2PO4, 2.9g Na2HPO4·12H2O, dissolved in 800mL pure water, pH adjusted to 7.2-7.4 with NaOH or HCl, and then brought to a final volume of 1000mL.

[0040] PBST: PBS containing 0.05% Tween 20;

[0041] Antibody dilution solution: PBS with 0.1% gelatin added.

[0042] TMB colorimetric solution: Solution A: 18.43g Na2HPO4·12H2O, 9.33g citric acid, diluted to 1000mL with pure water; Solution B: 60mg TMB dissolved in 100mL ethylene glycol. Mix solutions A and B in a 5:1 ratio to obtain the TMB colorimetric solution. Mix fresh before use.

[0043] Nitrosamine hapten: In this invention, 4-amino-3,5-dichlorobenzoic acid is selected as the nitrosamine hapten, and its molecular structure is as follows:

[0044]

[0045] The detection methods involved in the following embodiments are as follows:

[0046] Method for detecting chloronitrosamine inhibition rate: The optimal antigen and antibody concentrations for ic-ELISA were selected using a checkerboard assay. The antigen was diluted to 0.03, 0.1, 0.3, and 1 μg / mL with carbonate buffer (CBS), and the antibody was diluted to 0.03, 0.1, 0.3, and 1 μg / mL with antibody dilution buffer. After selecting the optimal operating point, the chloronitrosamine standard was diluted to concentrations of 0, 0.1, 0.3, 1, 3, 9, 27, and 81 ng / mL. Following the ic-ELISA procedure, the results were plotted using OriginPro 8.5 (see results below). Figure 1 (As shown), the standard inhibition curve of chloronitrosamine was obtained, and the IC50 was calculated. 50 .

[0047] Example 1: Synthesis of chloronitrosamine complete antigen

[0048] Weigh 5 mg of 4-amino-3,5-dichlorobenzoic acid and 8.28 mg of N-hydroxysuccinimide (NHS), dissolve them in 300 μL of N,N-dimethylformamide (DMF), and stir at room temperature for 10 min to obtain mixed solution A. Then weigh 13.75 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), dissolve it thoroughly in 100 μL of DMF, and add it to mixed solution A. Stir at room temperature for 4-6 h to obtain mixed solution B. Take 6 mg of hemocyanin (KLH), dilute it to 3 mg / mL with 0.01 M carbonate buffer (CBS), and slowly add mixed solution B dropwise to it. React at room temperature overnight to obtain the reaction solution. Then dialyze the reaction solution with 0.01 M PBS to remove unreacted small molecule haptens, obtaining the complete chloronitrosamine antigen BJS-KLH, which is identified by ultraviolet absorption scanning.

[0049] Example 2: Synthesis of chloronitrosamine-coated antigen

[0050] 3 mg of 4-amino-3,5-dichlorobenzoic acid and 4.97 mg of N-hydroxysuccinimide (NHS) were dissolved in 300 μL of anhydrous N,N-dimethylformamide (DMF) and stirred at room temperature for 10 min to obtain mixed solution C. 8.25 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was dissolved in 100 μL of anhydrous DMF and added to mixed solution C. The mixture was stirred at room temperature for 4-6 h to obtain mixed solution D. 6 mg of chicken ovalbumin (OVA) was diluted with 1 mL of 0.01 M carbonate buffer (CBS), and then mixed solution D was added dropwise to it to obtain the reaction solution. The reaction solution was dialyzed with PBS to remove unreacted small molecule haptens, yielding chloronitrosamine-coated antigen (BJS-OVA).

[0051] Example 3: Preparation of hybridoma cell lines secreting chloronitrosamine monoclonal antibodies

[0052] 1. Obtaining Animal Immunization: Complete Freund's adjuvant containing antigen was prepared by mixing nitramine complete antigen with an equal volume of complete Freund's adjuvant. Incomplete Freund's adjuvant containing antigen was prepared by mixing nitramine complete antigen with an equal volume of incomplete Freund's adjuvant. BALB / c mice were immunized by subcutaneous injection at multiple sites in the neck and back (except for sprint immunization). The initial immunization used complete Freund's adjuvant containing antigen at a dose of 100 μg / mouse. Multiple booster immunizations used incomplete Freund's adjuvant containing antigen at half the dose (50 μg / mouse). For sprint immunization, no adjuvant was used; the complete antigen diluted with physiological saline was injected intraperitoneally at a dose halved to 25 μg / mouse. The interval between the initial and second booster immunizations was one month, the interval between multiple booster immunizations was 21 days, and the interval between sprint immunization and the final booster immunization was 18-21 days. The immunization effect in mice was observed using an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA), i.e., the titer and inhibition of mouse serum were detected.

[0053] 2. Cell fusion: Three days after the sprint immunization, cell fusion was performed using the standard PEG (polyethylene glycol, molecular weight 4000) method. The specific steps are as follows:

[0054] a. After euthanizing the mouse by tail dislocation and cervical dislocation, immediately disinfect the mouse in 75% alcohol for about 5 minutes. Under aseptic conditions, remove the spleen and grind it moderately with the rubber tip of a syringe and pass it through a 200-mesh cell sieve to obtain a spleen cell suspension. Collect the suspension, centrifuge (1200 rpm, 8 minutes), wash the spleen cells three times with RPMI-1640 medium, and after the last centrifugation, dilute the spleen cells to a certain volume, count them, and set them aside for later use.

[0055] b. Collection of SP2 / 0 cells: 7-10 days before fusion, SP2 / 0 tumor cells are cultured in RPMI-1640 medium containing 10% FBS (fetal bovine serum) in a 5% CO2 incubator. Before fusion, the number of SP2 / 0 tumor cells per milliliter of medium should reach (1-4) × 10⁻⁶. 7 To ensure that SP2 / 0 tumor cells are in the logarithmic growth phase before fusion, tumor cells are collected during fusion, suspended in RPMI-1640 basal culture medium, and cell counting is performed.

[0056] c. Fusion process (7 min): Min 1, add 1 mL of PEG 1500 to the cells dropwise from slow to fast; Min 2, let stand; Min 3 and Min 4, add 1 mL of RPMI-1640 medium dropwise over 1 min; Min 5 and Min 6, add 2 mL of RPMI-1640 medium dropwise over 1 min; Min 7, add 1 mL of RPMI-1640 medium dropwise every 10 s; then incubate at 37°C for 5 min; centrifuge (800 rpm, 8 min), discard the supernatant, resuspend in RPMI-1640 selection medium containing 20% ​​fetal bovine serum and 2% 50×HAT, add 200 μL / well to a 96-well cell plate, and incubate at 37°C in a 5% CO2 incubator;

[0057] 3. Cell screening and cell line establishment: On the 3rd day of cell fusion, the fused cells were screened with RPMI-1640 medium with half medium replacement. On the 5th day, the medium was completely replaced with RPMI-1640 transition medium containing 20% ​​fetal bovine serum and 1% 100×HT. On the 7th day, the cell supernatant was collected for screening.

[0058] The screening process consists of two steps: First, positive cell wells are selected using ic-ELISA; second, chlornitramine is used as a standard, and the inhibitory effect on positive cells is determined using ic-ELISA.

[0059] Cell wells that showed good inhibition of chlornitramine standard were selected, and subcloning was performed using the limiting dilution method. The same method was used to detect the cells after 7 days.

[0060] Three subclonings were performed using the method described above to finally obtain a hybridoma cell line that secretes chloronitrosamine monoclonal antibody.

[0061] Example 4: Preparation and Identification of Chlornitramine Monoclonal Antibody

[0062] 8-10 week old BALB / c mice were injected intraperitoneally with 1 mL of sterile paraffin oil; 7 days later, each mouse was injected intraperitoneally with 1×10 6 Chlornitramine hybridoma cells were used to collect ascites fluid starting from day 7. The ascites fluid was then purified for antibody treatment using the caprylic acid-saturated ammonium sulfate method.

[0063] Under slightly acidic conditions, octanoic acid can precipitate other proteins in the ascites fluid besides IgG immunoglobulin. After centrifugation, the precipitate is discarded. Then, an equal volume of saturated ammonium sulfate solution is used to precipitate IgG-type monoclonal antibodies. After centrifugation, the supernatant is discarded. The antibodies are dissolved in 0.01M PBS solution (pH 7.4), dialyzed to desalt, and finally purified monoclonal antibodies are obtained and stored at -20℃.

[0064] Using an indirect competitive ELISA, nitrosamine was detected with a nitrosamine monoclonal antibody, and the IC50 of nitrosamine was determined. 50 The value was 3.5 ng / mL, indicating good detection sensitivity for nitrosamines. Simultaneously, the nitrosamine monoclonal antibody was used to detect nitrosamine analogs (2,4-dichloroaniline, 4,5-dichloro-2-nitroaniline, 2-chloro-5-nitroaniline, and 2-nitro-1,4-phenylenediamine), and the cross-contamination rate was less than 10% (IC50). 50 The concentration of the monoclonal antibody is >35 ng / mL, indicating that it has good specificity (see Table 1). Therefore, it can be used for the immunoassay detection of chloronitrosamine. (Cross-cross rate = (IC50 of chloronitrosamine)) 50 ICs of similar types 50 (×100%)

[0065] Table 1 Cross-reactivity rates of chlornitramine monoclonal antibodies

[0066] aniline compounds <![CDATA[IC 50 Value (ng / ml)]]> Cross-reactivity Chlornitramine 3.5 100％ 2,4-Dichloroaniline >35 ＜10％ 4,5-Dichloro-2-nitroaniline >35 ＜10％ 2-Chloro-5-nitroaniline >35 ＜10％ 2-Nitro-1,4-Phenylenediamine >35 ＜10％

[0067] Example 5: Application of chloronitrosamine monoclonal antibody

[0068] The monoclonal antibody prepared from hybridoma cell lines via in vivo ascites fluid was used in an ELISA addition and recovery assay for chloronitrosamine. The specific steps are as follows:

[0069] (1) Coat a 96-well microplate with 0.3 μg / mL of the coating stock diluted with carbonate buffer (CBS), 100 μL per well, and coat at 37℃ for 2 h. Then wash the plate three times with PBST washing buffer, 200 μL per well each time, for 3 min each time, and pat dry.

[0070] (2) Block with CBS containing 0.2% gelatin, 200 μL per well, block at 37°C for 2 h, wash the plate three times with PBST washing solution, 200 μL per well each time, 3 min each time, and pat dry;

[0071] (3) Prepare 0, 0.1, 0.3, 1, 3, 9, 27 and 81 ng / mL chloronitramine standard solutions with phosphate buffer (PBS). Add the standard solutions and the extracts of the samples to be tested to the sealed microplates, 50 μL per well, and repeat each sample in 3 wells. Then add 50 μL of anti-chloronitramine monoclonal antibody diluted 1:32000 to each well. After reacting at 37℃ for 0.5 h, wash the plate and pat dry.

[0072] (4) Add 100 μL of HRP-labeled goat anti-mouse IgG secondary antibody diluted 1:3000 with PBS containing 0.1% gelatin to each well, react at 37°C for 0.5 h, then wash and pat dry.

[0073] (5) Add 100 μL of TMB colorimetric solution to each well, develop the color at 37℃ for 15 min, then add 50 μL of 2M H2SO4 stop solution to each well, and measure the absorbance at 450 nm.

[0074] The standard curve of chloronitrosamine inhibition by monoclonal antibody is shown below. Figure 1 As shown, the IC50 of chlornitramine monoclonal antibody was determined by ic-ELISA. 50 The value was 3.5 ng / mL, indicating that the antibody has good sensitivity to chloronitrosamine and can be used for the immunoassay detection of chloronitrosamine.

[0075] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A hybridoma cell line that secretes monoclonal antibodies against chloronitrosamine, characterized in that, The hybridoma cell line has the accession number CGMCC No. 45917.

2. A chloronitrosamine monoclonal antibody, characterized in that, The chloronitrosamine monoclonal antibody is secreted by the hybridoma cell line described in claim 1.

3. A composition, characterized in that, The composition comprises the hybridoma cell line of claim 1 and / or the chloronitrosamine monoclonal antibody of claim 2.

4. A test strip, characterized in that, The test strip contains the hybridoma cell line of claim 1, the chloronitrosamine monoclonal antibody of claim 2, or the composition of claim 3.

5. A reagent kit, characterized in that, The kit contains the hybridoma cell line of claim 1, the chloronitrosamine monoclonal antibody of claim 2, or the composition of claim 3.

6. A chip, characterized in that, The chip contains the hybridoma cell line of claim 1, the chloronitrosamine monoclonal antibody of claim 2, or the composition of claim 3.

7. The use of the chloronitrosamine monoclonal antibody of claim 2, the composition of claim 3, the test strip of claim 4, the kit of claim 5, or the chip of claim 6 in the detection of chloronitrosamine.

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